Procedure to block the replication of reverse transcriptase dependent viruses by the use of inhibitors of deoxynucleotides synthesis

ABSTRACT

A method for inhibiting replication of reverse transcriptase dependent virus in plant or animal cells, comprising the step of administering to said cells a compound that depletes the intracellular pool of deoxyribonucleoside phosphate in an amount effective to inhibit replication of said virus. Hydroxyurea is one such suitable compound. Also disclosed is a method for producing incomplete reverse- transcriptase dependent viral DNA, by administering a deoxyribonucleoside phosphate-depleting drug to cells infected with such a virus.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 08/065,815, filed May 21, 1993.

FIELD OF THE INVENTION

[0002] The present invention relates generally to the field of reversetranscriptase dependent viruses. More specifically, the inventionrelates to the use of agents which reduce intracellular concentrationsof deoxyribonucleosides as a means to inhibit the replication of reversetranscriptase dependent viruses.

BACKGROUND OF THE INVENTION

[0003] Viruses are microorganisms that depend, to some degree, on hostcell components for their growth and replication. Viral infection andreplication in host cells generally results in disease, whether the hostis an animal or plant. Human diseases caused by viral infections includethe acquired immunodeficiency syndrome (AIDS) and hepatitis. A generaldiscussion of this field is presented in Fundamental Virology, SecondEdition, (ed. B. N. Fields, D. M. Knipe, R. M. Chanock, M. S. Hirsh, J.L. Melnick, T. P. Monath, and B. Roizman, Raven Press, Ltd., New York,N.Y. 1991).

[0004] Retrovirus Replication

[0005] Retroviruses comprise a large family of viruses that primarilyinfect vertebrates. Many diseases, including the induction of sometumors, are associated with retroviral infection (see FundamentalVirology, supra, pp. 645-708). All retroviruses, regardless of theirclinical manifestations, have related structures and modes ofreplication.

[0006] Retroviruses contain an RNA genome that is replicated through aDNA intermediate. Inside the cell, the viral genome serves as a templatefor the synthesis of a double-stranded deoxyribonucleic acid (DNA)molecule that subsequently integrates into the genome of the host cell.This integration occasionally results in the induction of a tumor in theinfected host organism. Following integration, a complex sequence ofevents leads to the production of progeny virions which are releasedfrom the infected cell.

[0007] Early in the retroviral life cycle, the RNA genome is copied intoDNA by the virally encoded reverse transcriptase (RT). This enzyme canuse both RNA and DNA templates, thereby producing the first strand ofDNA (the negative strand) from the infecting RNA genome and acomplementary second strand (the positive strand) of DNA using the firstDNA strand as a template. To synthesize these DNA strands, the RTutilizes cellular substrates called deoxynucleoside triphosphates(dNTP).

[0008] Human retroviruses can be grouped into the leukemia viruses (HTLVtype viruses) and the immunodeficiency viruses (HIV type viruses). HTLVinfection may lead to one form of leukemia. Acquired immunodeficiencysyndrome (AIDS) is caused by a form of HIV, with HIV-1 being morevirulent than HIV-2. Both HTLV and HIV infect peripheral bloodlymphocytes (PBL).

[0009] Other animal retroviruses include feline leukemia virus (FeLV)and lentiviruses. Virulent FeLV infection generally results in fatalaplastic anemia in cats. Lentiviruses cause a variety of neurologicaland immunological diseases such as visna in sheep and infectious anemiain horses.

[0010] HIV Infection

[0011] HIV-1 was first identified as the causative agent of AIDS in1983. The AIDS pandemic is now one of the most serious health problemsworldwide. Catastrophic medical and social consequences are likely toextend into the next century. The World Health Organization (WHO) hasestimated that between eight and ten million people are currentlyinfected with HIV, and that approximately ten times as many individualswill be affected in the next decade. The large pool of HIV carriersmakes the development of effective antiviral treatments a medicalpriority.

[0012] Hepatitis B Infection

[0013] Hepatitis B virus (HBV) is one of at least three (A, B and C)viruses that selectively infect liver cells (for a general discussion ofHBV see Fundamental Virology, supra, pp. 989-1021). HBV infections tendto be persistent with minimal liver damage or with chronic hepatitisthat may lead to cirrhosis or liver cancer (hepatocellular carcinoma orHCC). Worldwide, more than 200 million people infected with HBV.

[0014] Other Viruses

[0015] Several other viruses that infect humans, animals and plants alsodepend on reverse transcriptase for replication. These includeretroviruses such as the leukemia viruses known to exist in severalspecies, including HTLV-1 in humans, as well as reverse transcriptasedependent DNA viruses, such as the cauliflower mosaic virus (a plantvirus).

[0016] Antiviral Therapies

[0017] There is a critical need to develop effective drug treatments tocombat RT dependent viruses such as HIV. Such efforts were recentlyurged in the United Kingdom-Irish-French Concorde Trial conclusionswhich reported that the nucleoside analog zidovudine (AZT), a mainstayin the treatment of patients infected with HIV-1, failed to improve thesurvival or disease progression in asymptomatic patients. Othernucleoside analogs like didanosine (ddl) are currently under evaluation.The effects of ddl on disease progression and patient survival endpointshave not been adequately investigated. Non-competitive HIV-1 RTinhibitors and HIV-1 protease inhibitors have also been recentlydeveloped. Despite the high efficacy of these compounds, the initial invitro/in vivo testing has been characterized by the rapid onset ofvariants of HIV-1 resistant to these drugs (escape mutants). Despitehaving different antiviral activities and pharmacokinetics properties,the drugs mentioned here all directly target HIV-1 proteins.

[0018] Although this latter approach must be continued, we havedeveloped a different antiviral strategy that targets one or morecellular components that are required for the replication of reversetranscriptase dependent viruses.

SUMMARY OF THE INVENTION

[0019] The present invention is based on the discovery that drugs whichreduce the intracellular concentration of deoxynucleoside, phosphatesinhibit the replication of reverse transcriptase dependent viruses: Suchdrugs act either by inhibiting the intracellular synthesis ofdeoxynucleoside phosphates or by depleting the intracellular pool ofdeoxynucleoside phosphates. Viruses sensitive to growth inhibition bylimiting deoxynucleoside phosphates are retroviruses, including HIVwhich causes AIDS, hepatitis B virus, cauliflower mosaic virus, andother reverse transcriptase dependent viruses. As one example,hydroxyurea limits synthesis of the intracellular deoxynucleosidephosphates by inhibiting enzymatic activity of ribonucleoside reductase.Other compounds are known that similarly inhibit accumulation ofintracellular deoxynucleoside phosphates by this mechanism or byaffecting other biosynthetic steps that lead to production ofintracellular deoxynucleoside phosphates. Compounds that limitintracellular deoxynucleoside phosphates can be used in conjunction withantiviral nucleoside phosphate analogs, which are themselves therapeuticas competitive inhibitors of native nucleosides, to increase theeffectiveness of antiviral treatment. Compounds that depleteintracellular deoxynucleoside phosphates may be used as an alternativeto treatment with antiviral nucleoside phosphate analogs, especiallywhen a virus has become refractory to nucleoside analog treatment.

[0020] One aspect of the present invention is a method for inhibitingreplication of reverse transcriptase dependent virus in animal cells,comprising the step of administering to the cells a compound thatdepletes the intracellular pool of deoxyribonucleoside phosphate in anamount effective to inhibit replication of the virus. The virus can, forexample, be a retrovirus, or a reverse transcriptase-dependent DNAvirus. The deoxynucleoside phosphate depleting compound in oneembodiment is a deoxynucleotide synthesis inhibitor. In anotherembodiment, the deoxynucleoside phosphate depleting compound is aninhibitor of ribonucleotide reductase. One preferred compound ishydroxyurea.

[0021] The invention can be used on cells in vitro or in vivo. Invarious preferred embodiments, the animal is a mammal or a bird.Preferably, the animal is a human.

[0022] In one specific embodiment, the virus is the humanimmunodeficiency virus (HIV), such as HIV-1 or HIV-2, and the cells arehuman cells. In another specific embodiment, the virus is hepatitis Band the cells are human cells.

[0023] The method of the present invention may be practiced by depletingthe intracellular pool of deoxynucleoside phosphates to limit viralreplication by limiting the rate of DNA chain elongation. For example,AZT and dideoxynucleosides, such as ddl, ddC and 2′-fluorodideoxynucleosides, so limit viral replication. This can result inpremature termination of viral DNA synthesis to produce incomplete viralDNA.

[0024] Another aspect of the present invention is a method forinhibiting replication of reverse transcriptase dependent virus inanimal cells, comprising the steps of administering to the cells acompound that depletes the intracellular pool of deoxyribonucleosidephosphate, and coadministering to the cells antiviral nucleosidephosphate analogs which compete with the pool of deoxyribonucleosidephosphates. Preferred antiviral nucleoside phosphate analogs includeAZT, ddl, and ddC.

[0025] A different aspect of the invention relates to a method ofproducing incomplete viral DNA from reverse transcriptase dependentvirus in animal cells, comprising the step of administering to the cellsa compound that depletes the intracellular pool of deoxyribonucleosidephosphate in an amount effective to inhibit replication of the virus.

[0026] Finally, the invention includes a method for inhibitingreplication of reverse transcriptase dependent virus in plant cells,comprising the step of administering to the cells a compound thatdepletes the intracellular pool of deoxyribonucleoside phosphate in anamount effective to inhibit replication of the virus.

BRIEF DESCRIPTION OF THE FIGURES

[0027]FIG. 1a graphically depicts p24 expression in HIV-1 infected PBLas a function of the hydroxyurea and ddl concentrations.

[0028]FIG. 1b graphically depicts the number of viable PBL in an HIV-1infected culture as a function of the hydroxyurea and ddlconcentrations.

[0029]FIG. 1c shows HIV-1 p24 expression normalized to the number ofviable cells as a function of the Hu and ddl concentrations.

[0030]FIG. 2 graphically depicts p24 expression in HIV-1 infected humanprimary macrophages as a function of hydroxyurea and AZT concentrations.

[0031]FIG. 3a graphically depicts a time course of p24 inhibition byhydroxyurea and/or by ddl in activated PBL isolated from an HIV-1infected patient.

[0032]FIG. 3b graphically depicts the number of viable cells isolatedfrom an HIV-1 infected patient that survived in culture with treatmentby hydroxyurea and/or ddl.

DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention is based on the discovery that a reductionof the intracellular deoxynucleoside triphosphate (dNTP) concentrationselectively inhibits the replication of reverse transcriptase dependentviruses. An approach to virus inhibition that is based on this strategyadvantageously avoids triggering the formation of viral escape mutants.Conversely, direct selective pressure against viral proteins would beexpected to promote the formation of such mutants.

[0034] In the practice of the present invention, hydroxyurea is onepreferred compound that depletes intracellular dNTP levels. Thiscompound is one of many inhibitors of ribonucleotide reductase, anenzyme catalyzing the reduction of ribonucleoside diphosphates to theirdeoxyribonucleoside counterparts for DNA synthesis. Other ribonucleotidereductase inhibitors include guanazole, 3,4-dihydroxybenzo-hydroxamicacid, N,3,4,5-tetrahydroxybenzimidamide HCl, 3,4-dihydroxybenzamidoximeHCl, 5-hydroxy-2-formylpyridine thiosemicarbazones, andα-(N)-heterocyclic carboxaldehyde thiosemicarbazones,4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone,N-hydroxy-N′-amino-guanidine (HAG) derivatives,5-methyl-4-aminoisoquinoline thiosemicarbazone, diaziquone, doxorubicin,2,3-dihydroxylbenzoyl-dipeptides and 3,4-dihydroxylbenzoyl-dipeptides,iron-complexed 2-acetylpyridine5-[(2-chloroanilino)-thiocarbonyl]-thiocarbonohydrazone (348U87),iron-complexed2-acetylpyridine-5-[(dimethylamino)thiocarbonyl]-thiocarbonohydrazone(A1110U), 2′-deoxy-2′-methylenecytidine 5′-diphosphate (MdCDP) and2′-deoxy-2′, 2′-difluorocytidine5′-diphospahtefdFdCDP),2-chloro-9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-adenosine(Cl-F-ara-A), diethyidithiocarbamate (DDC),2,2′-bipyridyl-6-carbothioamide, phosphonylmethyl ethers of acyclicnucleoside analogs, [eg. diphosphates ofN-(S)-(3-hydroxy-2-phosphonylmethoxypropyl andN-2-phosphonylmethoxyethyl) derivatives of purine and pyrimidine bases],nitrosourea compounds, acylclonucleoside hydroxamic acids (e.g.,N-hydroxy-α-(2-hydroxyethoxy)-1 (2H)- pyrimidineacetamides 1-3, and2-acetylpyridine 4-(2-morpholinoethyl)thio-semicarbazone (A723U)).

[0035] Compounds that inhibit dNTP synthesis or that otherwise depletethe intracellular pool of at least one dNTP may be administered by anyconventional route. Where treated cells are in vitro, the compound maysimply be introduced into the medium in which the cells are growing. Onthe other hand, where cells to be treated are part of a larger organism,that is, where treatment is in vivo, administration to an animal may bevia the oral route, or may be intravenous, intraperitoneal,intramuscular, subcutaneous, transdermal, transmucosal (e.g., byinhalation or by means of a suppository), or by any other suitableroute. Administration to plants may be accomplished by spraying,dusting, application in irrigation water, or by any other conventionalmeans.

[0036] It should be noted that depletion of the intracellular pool ofany one of the four deoxynucleoside phosphates is considered to bewithin the scope of the present invention. Furthermore, depletion ofmono-, di-, or triphosphates of nucleosides is also within the scope ofthis invention.

[0037] The particular dosage, toxicity, and mechanism for delivery ofthe dNTP-depleting drugs of the present invention are either alreadyknown, or can be readily determined by conventional empiricaltechniques. Although some of the dNTP-depleting compounds may exhibitlimiting toxicity or difficulties in intracellular delivery, others(such as hydroxyurea) have been extensively studied and found to havefavorable pharmacological properties.

[0038] Suitable human dosages for these compounds can vary widely.However, such dosages can readily be determined by those of skill in theart. For example, dosages to adult humans of from about 0.1 mg to about1 g or even 10 g are contemplated.

[0039] In one preferred embodiment, the dosage is such that theintracellular dNTP pool is depleted to a concentration that is below theK_(m) of the viral reverse transcriptase, but above the K_(m) ofendogenous cellular polymerases, such as DNA polymerase α, β, and γ.This permits selective inhibition of viral replication withoutsignificant cellujar toxicity.

[0040] Hydroxyurea has been widely used in cancer therapy as a broadspectrum antineoplastic drug (R. C. Donehower, Seminars in Oncology 19(Suppl. 9), 11 (1992)). Hydroxyurea is readily absorbed after oralingestion, rapidly distributed in the body fluids, including thecerebrospinal fluid, and enters cells efficiently by passive diffusion(Id.). Its toxic effects are less profound and easier to control thanother chemotherapeutic drugs (Id.).

[0041] In human chemotherapy, hydroxyurea is currently administeredusing two basic schedules: (a) a continuous daily oral dose of 20-40 mgper kg per day, or (b) an intermittent dose of 80 mg per kg per everythird day. Either schedule could be used in the treatment of viralinfections. However, because response to treatment is variable,peripheral white blood cell counts must be monitored so that treatmentcan be stopped when leukopenia occurs. Similar dosage ranges may be usedin the practice of the present invention.

[0042] Given that viral reverse transcriptase is generally quitesensitive to decreased levels of dNTP, lower dosages of hydroxyurea mayalso be effective in treating viral infections. Such low dosages ofhydroxyurea would reduce the toxicity to white blood cells. Any dosagethat effectively decreases the replication of RT-dependent viruses wouldbe useful in chronically treating AIDS patients.

[0043] In the practice of the present invention, the inhibition ofreverse transcriptase activity and the impairment of HIV-1 DNA synthesisare accomplished by treating cells with hydroxyurea. Under the specifiedconditions, incomplete HIV-1 DNA was formed without apparent toxiceffects to the cells. Incomplete viral DNA has been shown to be rapidlydegraded in PBL (Zack, et al., supra, 1990 and 1992). Therefore, thepresent invention provides a new method to inhibit HIV replication bymodulating intracellular dNTP pools. This is accomplished by employingdrugs such as hydroxyurea at pharmacological ranges.

[0044] The present invention also encompasses antiviral therapies thatare based on the use of dNTP-depleting drugs in conjunction withconventional or novel nucleoside phosphate analogs. By depleting theintracellular dNTP pool, drugs such as hydroxyurea are expected toincrease the therapeutic effect of treatment of HIV infection bynucleoside phosphate analogs such as AZT, ddl, ddC, 2′-F-dd-ara-A,2′-F-dd-ara-I and 2′-F-dd-ara-G. These analogs act as competitors ofcellular dNTP according to an antiviral mechanism that is distinct fromthat of hydroxyurea. A description of the 2′-fluoro nucleosides has beenpresented by Marquez et al. in J. Med. Chem. 33:978-985 (1990).Currently, antiviral therapy requires doses of AZT or ddl at 500 mg perday or ddC at 2 mg per day for an adult human. Similar dosages may beused in the present invention. However, use of dNTP depleting drugs mayincrease the effectiveness of these nucleoside phosphate analogs so thatthey can be used at lower dosages or less frequently.

[0045] One of the problems in using antiviral nucleoside phosphateanalogs is the appearance of escape mutants. Such variants usuallyderive from mutations in the gene that encodes RT. We believe theappearance of RT mutants that can function using low levels ofnucleotides will be an unlikely event. Hence, we believe that antiviraldrugs, such as hydroxyurea, which deplete intracellular dNTP pools willbe unlikely to favor the evolution of RT escape mutants. Furthermore,drugs that deplete the intracellular dNTP pool could be of value in thetreatment of viral disease in cases where RT escape mutants haveappeared.

[0046] Because dNTP-depleting drugs and nucleoside phosphate analogshave different inhibitory mechanisms, we predict that combinations ofthese agents will result in synergistic inhibitory effects. By depletingthe intracellular nucleotide pool with hydroxyurea or a similarly actingdrug, the therapeutic effects of nucleoside phosphate analogs, which actas competitors of dNTP, are expected to increase. Such a combinationdrug treatment may also result in decreased toxicity since lower dosagesof nucleoside phosphate analogs would be rendered more effective.

[0047] Because HIV-1 RT is a distributive enzyme, we expected that lowlevels of dNTPs induced by drugs such as hydroxyurea would affect RTmore than the cellular DNA polymerases α, β, and γ, which are known tobe processive enzymes (Huber, et al., supra; Kati, et aL, supra; U.Hübscher, supra). This selective effect on RT may result in lowercellular toxic effects than occur with other antiviral drugs.

[0048] Unlike retroviruses, HBV is a DNA virus with a partiallydouble-stranded and partially single-stranded genome. However, likeretroviruses, reverse transcription is required early in the process ofHBV genome replication. The RT is specified by the HBV genome andsynthesized in the infected host liver cell where viral replicationoccurs.

[0049] Because replication of the HBV viral genome is dependent on RT,it is expected that the method of limiting dNTP pools by treating peoplewith therapeutic drugs that inhibit dNTP synthesis would also beeffective in limiting HBV viral replication. Drugs such as hydroxyureathat diffuse into nearly all cell types would be particularlyadvantageous in controlling hepatic replication of HBV.

[0050] Limiting HBV replication has two important effects. First, itlimits the spread of infectious virions from carriers to uninfectedindividuals. Second, it decreases the symptoms such as chronic hepatitisin infected individuals. Generally, liver function improves after HBVreplication ceases. Also, because the incidence of HCC is much higher inHBV-infected humans, decreased infection in the population presumablywould result in a decreased incidence of liver cancer.

[0051] As described above, the use of hydroxyurea (or similardNTP-limiting drugs) in conjunction with antiviral drugs, such asadenine arabinoside, ara-monophosphate, acyclovir, 6-deoxyacyclovir, andα, β, and γ interferons, that act via other mechanisms could alsoincrease the effectiveness of these anti-HBV drugs. This is especiallypredicted for adenine arabinoside which acts as a competitive inhibitorin a mechanism analogous to that of antiviral nucleoside phosphateanalogs used to treat HIV infections. Furthermore, treatment withhydroxyurea (or similar dNTP-limiting drugs) could make antiviral drugsmore effective at lower doses than required for treatment solely usingantiviral drugs.

[0052] As described above, the method of using hydroxyurea (or similardNTP-limiting drugs) on people whose HBV infections have becomerefractory to antiviral drugs is also anticipated in the presentinvention.

[0053] Other viruses that infect animals or plants are also dependent onRT activity for their replication. Cauliflower mosaic virus is oneexample of a virus that uses a RT in replication of its DNA genome.

[0054] The botanical use of compounds that limit intracellular dNTPpools to inhibit viral replication of other reverse transcriptasedependent viruses is within the scope of the present invention, as isthe use of such compounds on animals, including humans, infected with awide variety of RT-dependent viruses.

[0055] The rationale for the present invention and the practice of thepresent invention may be better understood by reference to the followingnonlimiting examples. ,

[0056] A key step of HIV-1 infection of PBL is the conversion of theviral RNA genome into double-stranded DNA by the action of HIV-1 RT.Viral DNA synthesis differs in different states of infected PBL. Inquiescent PBL, viral DNA synthesis can be initiated as efficiently as inmitogen-stimulated PBL. However, in contrast to the stimulated cells,DNA synthesis in quiescent PBL may terminate prematurely (J. A. Zack, etal., Cell 61:213 (1990); J. A. Zack, et al., Virology 66:1717 (1992))producing no HIV-1 progeny (Zack, et al, supra; M. Stevenson, et al.,EMBO J. 9:1551 (1990); M. I. Bukrinsky, et al., Science 254:423 (1991))This process results in a pool of unintegrated viral DNA (Stevenson, etal., supra; Bukrinsky, et al., supra), which can remain latent in bothin vitro infected quiescent PBL and in vivo infected resting PBL (Zack,et al., supra, 1990 & 1991; Stevenson, et al., supra; Bukrinsky, et al.,supra). Stimulation of these cells can rescue HIV-1 DNA, leading tointegration and production of viral progeny (Id.). Incomplete viral DNAhas also been found associated with HIV-1 mature infectious particles,but the biological role of this DNA is unclear (F. Lori, et al., J.Virol. 66:5067 (1992); D. Trono ibid. 66:4893 (1992)).

[0057] Example 1 illustrates a method that can be used to quantitate thereplication of the HIV-1 genome in infected cells. In this example, therates of HIV-1 DNA synthesis in infected quiescent and stimulated PBLwere quantitatively analyzed using a polymerase chain reaction (PCR)assay.

EXAMPLE 1 HIV Replication

[0058] The PCR assay, previously applied to quantitate HIV-1 DNA inmature HIV-1 virions (F. Lori et al., supra; D. Trono, supra), was usedto amplify several regions of the HIV-1 genome. The primer pairs used toamplify the viral DNA were M667/AA55, M667/BB301, and M667/M661 (M.Stevenson et al., supra; M. 1. Bukrinsky, et al., supra). M667 is asense primer in the R region of the long terminal repeat (LTR). AA55 isan antisense primer immediately 5′ to the PB (tRNA primer binding)region. The M667/AA55 primer pair amplifies the negative strand regioninitially synthesized by RT to yield a product called R-U5. BB301 iscomplementary to the PB region. Amplification by M667/BB301 can beachieved in the presence of positive stand DNA which has beensynthesized starting at the polypurine tract upstream from the right LTRand, after jumping to the other end of the template, extended up to thePB region to yield a product called R-PB (H. E. Varmus and R. Swanstrom,in Replication of Retroviruses, RNA Tumor Viruses. R. Weiss, N. Teich,H. Varmus, J. Coffin, Eds. (Cold Spring Harbor Laboratory, Cold SpringHarbor, 1984), pp. 369-512). The negative strand, which is not fullycompleted, is not expected to be amplified because the RNA sequenceswhich are complementary to the PB region have been digested in theseexperiments. M661 is an antisense primer in the gag region.Amplification by M667/M661 reflects the presence of complete negativestrand DNA to yield a product called R-gag. These primers were designedto estimate the extent of reverse transcription at three differentreplicative steps: R-U5, initial negative strand synthesis; R-PB,initial positive strand synthesis up to the tRNA primer binding region;and R-gag, complete negative strand synthesis. These steps occur insubsequent order during reverse transcription (Varmus and Swanstrom,supra). If the DNA carried by the virus was a full-length negativestrand DNA, the three regions analyzed by quantitative PCR should beamplified to equivalent levels. β globin sequences were amplified fromthe same DNA extracts in order to normalize the amount of DNA used asdescribed in J. A. Zack et al., supra (1990); and J. A. Zack et al.,supra (1 992).

[0059] Viral DNA was detected immediately after infection of quiescentPBL and the amount of DNA observed at that time was proportional to theinitial multiplicity of infection (MOI) of the HIV-1 IIIB strain (M.Popovic, et al., Science 224:497 (1984)). MOI of 1 and 10 were used andviral DNA was detected comparable to HIV-1 DNA standards correspondingto about 100 and 1000 copies, respectively, of HXB2(RIP7) plasmid DNA(J. M. McCune, et al., Cell 53:55 (1988)) for the R-U5, R-PB and R-gagregions.

[0060] This DNA was incompletely replicated, the typical form associatedwith the mature HIV-1 particles (F. Lori et al., supra; D. Trono supra).These results suggest that a portion of the incomplete DNA observed inPBL at early phases of infection was contributed by the DNA carried bythe infectious viruses. Viral DNA synthesis for 72 hours after infectionwas next analyzed. HIV-1 DNA synthesis in quiescent PBL wassignificantly slower and less efficient than in stimulated PBL. Inparticular, in quiescent PBL the initial synthesis of viral DNA at theorigin of retroviral DNA replication (immediately upstream to the tRNAprimer binding region, represented by the R-U5 product of the PCRreaction) was achieved relatively early after infection (after 10hours), while the completion of full-length negative strand DNAsynthesis was significantly delayed (between 48 and 72 hourspost-infection, represented by the R-gag product of the PCR assay). Incontrast, synthesis of full-length negative strand DNA was completedwithin 10 hours after infection in stimulated PBL.

[0061] Moreover, in stimulated PBL the DNA synthesis progressivelyincreased during the time course at much higher levels than in quiescentPBL.

[0062] In summary, we found the total amount of viral DNA produced inquiescent PBL was significantly less than that produced in stimulatedPBL. Even at 72 hours, the amount of viral DNA in quiescent PLB wasabout 10-fold less than the amount produced in stimulated PBL after 10hours of growth. After 72 hours of growth, the total amount of viral DNAproduced in stimulated PBL was at least 100-fold more than the amountproduced in quiescent PBL.

[0063] Conflicting observations have been reported previously regardingthe form of HIV-1 DNA in infected quiescent lymphocytes. An incompleteDNA in infected quiescent cells was reported by Zack et al. (supra, 1990and 1992). On the other hand, Stevenson et al. (supra) showed latentcomplete DNA was present in quiescent PBL, but this DNA wasunintegrated. These discrepancies could be explained by our findingsthat DNA synthesis proceeds in a slow and inefficient manner inquiescent PBL.

[0064] Previous studies have shown that cellular enzymes which areresponsible for dNTP synthesis, such as thymidine kinase anddeoxycytidine kinase, have extremely low activities in quiescent PBL,that increase dramatically in activated PBL (L. Pegoraro and M. G.Bernengo, Exp. Cell Res. 68:283 (1971)). Low levels of dNTP synthesisand the high turnover rate of dNTP during DNA replication (J. Ji and C.K. Mathews, Mol. Gen. Genet. 226:257 (1991)) would deplete theintracellular dNTP pool. In steady-state kinetics, if the dNTP poolswere significantly lower than the Michaelis constant, K_(m), most of thecatalytic potential of HIV-1 RT would be wasted and the rate of theviral DNA synthesis would be expected to be very sensitive to changes indNTP concentrations (I. H. Segel, in Biochemical Calculations (JohnWiley & Sons, New York, 1975)).

[0065] Example 2 illustrates the correlation between the low levels ofdNTP in quiescent PBL and the low rate of viral DNA synthesis that wasdescribed above.

EXAMPLE 2 Correlation Between dNTP Pool and HIV Replication

[0066] PBLs were cultured in the presence or absence ofphytohemagglutinin A (PHA) at 10 μg/ml for 48 hours. Intracellular dNTPwere extracted with 60% methanol and were examined by an enzyme assayusing synthetic oligonucleotides (P. A. Sherman and A. J. Fyfe, Anal.Biochem. 180:222 (1989)). Data represent the mean value of threeexperiments. K_(m) values were determined using a 600-base globin mRNAas template and were 3.8, 4.0, 3.9, and 2.6 μM for dCTP, dTTP, dGTP, anddATP, respectively. The cellular volume of PBL was measured using aCoulter counter chanalizer and found to be approximately 0.25 μl/10⁶cells for quiescent PBL and 0.38 μl/10⁶ cells for stimulated PBL,respectively.

[0067] As shown in Table 1, the levels of dNTP in quiescent PBL weresignificantly lower than in the stimulated PBL. The latter weresignificantly higher than the K_(m) of HIV-1 RT. Similar results wereobtained after infection with HIV-1. TABLE 1 Comparison ofdeoxyribonucleoside triphosphate pools (μM) in quiescent and PHAstimulated PBL cells. Treatment dATP dGTP dCTP dTTP PBL 0.32 ± 0.04 0.52± 0.12  1.48 ± 0.40  5.60 ± 0.80 PBL + PHA 3.24 ± 0.08 8.00 ± 2.67 18.13± 1.86 26.13 ± 1.60

[0068] We also assessed the in vitro activity of recombinant HIV-1 RT atdNTP concentrations that were equivalent to those found in quiescent andstimulated PBL. DNA was synthesized using a globin mRNA template and anoligo dT₁₆ primer (a primer extension assay). The HIV-1 RT reactionmixture contained 50 mM Tris-HCI (pH 8.0), 6 mM MgCl₂, 76 mM KCl, 0.5 mMDTT, 80 nM globin mRNA primed with oligo dT₁₆ in 1:5 ratio, and dNTP at(a) the concentrations equivalent to quiescent cells and (b) theconcentrations equivalent to stimulated cells as described in Table 1.Recombinant HIV-1 RT (obtained from American Biotechnologies) was usedat 5 U/ml.

[0069] Under the nucleotide concentrations that characterized quiescentconditions, the rate and yield of total DNA synthesis were profoundlylower than those corresponding to the stimulated condition. The rates ofdTMP incorporation by HIV-1 RT for quiescent conditions and stimulatedconditions are presented in Table 2. This could explain why DNAsynthesis was slower and less efficient in quiescent than in stimulatedPBL. TABLE 2 Rates of dTMP incorporation in vitro (ρmol per unit ofHIV-1 RT) in quiescent (−PHA) and stimulated (+PHA) PBL Incubation Time(min) PBL 0 30 60 90 120 −PHA 0 1.2 ± 0.1 4.2 ± 0.8 7.6 ± 0.8 12.8 ± 1.6+PHA 0 21.6 ± 2.2  49.6 ± 5.8  104.4 ± 13.6  153.0 ± 10.6 ratio of 18.011.8 13.7 12.0 +PHA/ −PHA

[0070] The mode of action of HIV-1 RT and the size of the DNA productswere further examined using the primer extension assay described aboveexcept that the template-primer was a 600-base globin mRNA primed witholigo(dT)₁₆that was labeled at the 5′ end. Aliquots were harvested at 0,15, 30, 60, and 120 minutes. Reaction products were separated by (a) 15%and (b) 6% polyacrylamide gel electrophoresis.

[0071] Two types of HIV RT activities were evident: an initialdistributive activity and a later processive activity. In the initialdistributive phase, the RT often became dissociated after incorporationof a dNTP into the nascent chain, giving rise to discrete molecularweight DNA products. This was particularly evident at dNTPconcentrations characteristic of quiescent PBL. In the gel lanes, thisgave rise to the ladder appearance of products ranging in size from the1 6-mer primer (at time 0) to approximately a 70-mer (after 60 minutesunder quiescent conditions). After 1 20 minutes incubation at quiescentPBL conditions, the longest DNA products measured approximately 70-100nt. After approximately 70 new nucleosides (nt) were added, theprocessivity of HIV-1 RT increased and higher molecular weight DNA wassynthesized. Processivity was observed primarily at dNTP concentrationssimilar to those in stimulated PBL. Processivity was seen after 15minutes incubation under stimulated PBL conditions resulting in DNAproducts over 70-100 nt; and it continued throughout the experimentresulting in full-length transcripts after 120 minutes incubation. Incontrast, little or no processivity was seen under quiescent PBLconditions, even after 120 minutes of incubation. These results suggestthat low concentrations of endogenous dNTP alone are sufficient toexplain the impaired DNA elongation observed in quiescent PBL.

[0072] This biphasic pattern of HIV-1 RT activity is in agreement withthe enzyme kinetics studies from others (H. E. Huber et al., J. Biol.Chem. 264:4669 (1989); W. M. Kati, K. A. Johnson, L. F. Jirva, K. S.Anderson, ibid. 267:25988 (1992)) and differs from the action of most ofthe replicative DNA polymerases which are processive polymerases, suchas E. coil pol I and IlIl, HSV DNA polymerase, and mammalian DNApolymerases a and y (Huber, et al., supra; Kati, et aL, supra; U.Hubscher, Experientia 39:1 (1983)).

[0073] Example 3 illustrates both that hydroxyurea can be used todeplete the intracellular dNTP concentration, and that such suboptimalconcentrations of dNTP cause incomplete HIV-1 DNA synthesis in PBL. The1 mM hydroxyurea concentration used in these procedures approximates theblood concentration of this drug during standard clinical protocols inhumans (R. C. Donehower, Seminars in Oncology 19 (Suppl. 9), 11 (1992)).Notably, hydroxyurea did not directly inhibit RT enzymatic activity evenat a 200-fold higher concentration (200 mM).

EXAMPLE 3

[0074] Use of Hydroxyurea to Inhibit HIV Replication

[0075] HIV-1 DNA synthesis was measured after infection of mitogen (PHA)stimulated PBL in the presence or absence of hydroxyurea. After 48 hoursof PHA stimulation and 24 hours pretreatment with 1 mM hydroxyurea,cells were infected with HIV-1 IIIB (Popovic et al., supra) in thepresence of hydroxyurea. Control cells were treated similarly, buthydroxyurea treatment was omitted. Cell aliquots were harvested 24, 48and 72 hours after infection and analyzed for the rate of dNTP synthesisinhibition (Sherman and Fyfe, supra) measured as the percentage of dNTPlevels compared to the control cells.

[0076] The results of this study, illustrated in Table 3, show that dNTPpools were substantially depleted in stimulated PBL incubated in thepresence of 1 mM hydroxyurea. TABLE 3 Effect of hydroxyurea on dNTPpools in treated PBL relative to untreated control PBL (% of untreatedcontrol amount). Incubation Time (hours) dNTP 0 24 48 72 dATP 100 ± 1019 ± 2 19 ± 1   7 ± 0.5 dGTP 100 ± 10 70 ± 5 45 ± 3 32 ± 3 dCTP 100 ± 1082 ± 9 61 ± 6 35 ± 4 dTTP 100 ± 10 115 ± 10 34 ± 2 23 ± 3

[0077] In addition, HIV-1 DNA synthesis was measured after infection ofPHA-stimulated PBL in the presence or absence of hydroxyurea using thePCR analysis as described above. Standards used for comparison wereserial dilutions of HXB2(RIP7) plasmid DNA (number of copies; McCune etal., supra) and f, globin DNA (nanograms).

[0078] Depletion of dNTP significantly affected the HIV-1 DNA synthesisrate and inhibited the completion of viral DNA synthesis in stimulatedPBL. Furthermore, dNTP depletion delayed production of full-lengthnegative strand viral DNA which was seen in only limited amounts(approximately 10-fold to 100-fold less over a 72 hour period) relativeto cells that were not treated with hydroxyurea. The pattern ofinhibition was quite similar to that observed in quiescent PBL. After 72hours, cell viability was comparable between hydroxyurea treated anduntreated cells.

[0079] Because most circulating lymphocytes in vivo are quiescent, therelevance of the population of quiescent infected PBL serving as areservoir of inducible HIV-1 has been recognized (Zack, et al., supra,1990 and 1992; Stevenson, et al., supra; Bukrinsky, et al., supra). Thelatent viral DNA pool in these cells clearly plays a role in viralrescue after mitogenic stimulation (Id.). Our results suggest amechanism of inefficient reverse transcription and subsequent formationof latent HIV-1 DNA in quiescent PBL. Although other mechanisms may alsoblock HIV-1 replication in quiescent PBL, naturally occurring low levelsof dNTP are sufficient to inhibit reverse transcription. ,

[0080] Example 4 illustrates that treatment of target cells withhydroxyurea, alone or in combination with the nucleoside phosphateanalog ddl, inhibits HIV-1 viral expression. In particular, viralexpression of RT in HIV-1 infected PHA-stimulated PBL was significantlyreduced by pre-infection treatment with 1 mM hydroxyurea.

EXAMPLE 4 Use of Hydroxyurea and/or ddl to Inhibit HIV Expression

[0081] PBL were stimulated with PHA for 48 hours and treated with 1 mMhydroxyurea for 24 hours prior to infection with HIV-1 IIIB (Popovic,etal., supra) in the presence of- the drug. Control cells were treatedsimilarly except that hydroxyurea treatment was omitted. Cellsupernatant aliquots were harvested two, five and nine days afterinfection and assayed for RT activity. RT activity was monitored as inExample 2. The results of this procedure are presented in Table 4. TABLE4 Inhibition of HIV-1 RT expression in infected PBL treated with 1 mMhydroxyurea (HU) RT Activity (cpm/ml × 1000) at Days Treatment 2 5 9 +HU0.892 0.589 0.434 −HU control 0.863 3.951 81.263

[0082] TABLE 5 Viral expression of p24 protein after HIV-1 infection ofPHA-stimulated PBL in the presence of μM of hydroxyurea (HU) and/or ddl(ddl). p24 (ng/ml) after infection (days) Treatment 4 8 12 none(control) 5 142 195 ddl 0.2 4 116 197 1 3 90 196 5 1 58 125 20 1 25 51HU 10 4 100 204 50 3 113 148 100 2 111 116 HU + ddl 10 + 0.2 3 144 20010 + 1 2 95 158 10 + 5 2 74 127 10 + 20 0 19 44 50 + 0.2 2 109 146 50 +1 1 70 128 50 + 5 0 12 24 50 + 20 0 0 0 100 + 0.2 2 105 95 100 + 1 0 5571 100 + 5 0 2 4 100 + 20 0 0 0

[0083] Furthermore, the hydroxyurea and ddl synergistically inhibitedHIV-1 p24 expression. That is, p24 expression decreased more when cellswere treated with both drugs rather than one or the other drug alone.For example, Table 5 shows that treatment of cells with 5 μM ddl yielded125 ng of p24 per ml of cell supernatant after 12 days of infection,while treatment with 50 μM hydroxyurea yielded 148 ng/ml after 12 daysof infection. However, when cells were treated with both 5 μM ddl and 50μM hydroxyurea, only 24 ng/ml of p24 were detected at day 12 afterinfection. Because of this synergistic effect, lower concentrations ofhydroxyurea and ddl in combination could effectively eliminate p24expression compared to treatment with only hydroxyurea or ddl at thesame concentrations.

[0084] Whereas the procedure in Example 4 involved the pre-treatment oftarget cells with hydroxyurea and/or ddl before infection, we alsoinvestigated the effect of drug treatment in cells that were alreadyinfected with the HIV-1 IIIB virus. In the latter procedure, we measuredp24 production during the course of an in vitro infection to assess theinhibition of HIV-1 replication in activated PBL.

[0085] Example 5 illustrates how hydroxyurea, either alone or incombination with ddl effects the production of p24 in HIV-1 IIIBinfected cells.

EXAMPLE 5 Effect of Hydroxyurea on P²⁴ Production

[0086] PBL from healthy donors were infected for 2 hour at 37° C. withHIV-1 [HTLV-IIIB] (m.o.i. =1) after 2 days stimulation with PHA andInterleukin-2 (IL-2). After washing out the residual virus, cells weretreated with hydroxyurea and/or ddl at the concentrations indicated(supernatant with no drug was used as control). Every 3-4 days,supernatant was harvested for p24 analysis, cells were counted and freshsupernatant and drugs were added. Samples were analyzed for (a) p24production in the supernatant, (b) count of viable cells, and (c) ratiosbetween the values expressed in (a)xand (b). The results from thisprocedure are presented in FIGS. 1a-1 c.

[0087] As shown in FIG. 1a, when used alone at low concentrations, or incombination with ddl, HIV-1 replication was inhibited in adose-dependent manner.

[0088] Notably, the combination of hydroxyurea and ddl completelyblocked HIV-1 replication (>99.9%), thus illustrating the powerfulsynergistic effect of this drug combination. Cell toxicity analysisreflected the known properties of the two drugs.

[0089] Hydroxyurea is known to act mainly as a cytostatic drug. However,continuous drug exposure eventually results in some cytotoxic effects(Yarbro, J. W., Semin. Oncol. 19:1 (1992)). This was also observed inour experiments at 0.1 mM hydroxyurea concentrations (FIG. 1b). However,both cytostatic and cytotoxic effects virtually disappeared when thedrug concentration was decreased (FIG. 1b).

[0090] Cytotoxicity of ddl is known to be low at the doses used in ourexperiments, which correspond to the plasma concentrations observed inAIDS treated patients (Faulds, D. and Brogden, R. N. Drugs 44:94(1992)). The combination of the two drugs did not significantly changethe cytotoxicity compared to the use of hydroxyurea alone.

[0091] This represents a further advantage of the hydroxyurea/ddlcombination since the antiviral effects were synergistically augmentedwithout a significant increase in cytotoxicity. To understand whetherthe different number of viable cells observed at different drugconcentrations or during the course of infection could have affected ourdata (less cells alive yielding less virus production), we normalizedthe p24 expression to the number of viable cells (FIG. 1c). Our resultsshowed the antiviral effect of hydroxyurea at low concentrations is notmediated by cytotoxicity.

[0092] We also investigated the inhibitory effects of hydroxyurea,either alone or in combination with AZT, on HIV-1 infection of primaryhuman macrophages. Although we found greater variability among differentexperiments that employed macrophages compared to our results withprimary PBL (note that in each experiment the same donor was used as asource of both PBL and macrophages), the dose-dependent inhibition ofHIV-1 production by hydroxyurea was nonetheless consistently more potentthan with primary PBL.

[0093] Example 6 illustrates the effectiveness of hydroxyurea as aninhibitor of HIV infection in macrophages. Moreover, this exampleillustrates the powerful synergistic effect of hydroxyurea and AZT asinhibitors of HIV infection of macrophages.

EXAMPLE 6 Time Course of HIV-1 Inhibition by Hydroxyurea and/or AZT inMacrophages

[0094] Macrophages were obtained by cell adhesion after purification ofPBL from healthy donors. After 14 days treatment withgranulocyte-macrophage-colony-stimulating factor, cells were infectedovernight with the HIV-1 strain Ba-L (Gartner et al., Science 233:215(1986)). Cells were subsequently washed and treated with hydroxyureaand/or with AZT at the indicated concentrations. Supernatants wereharvested ever 4-5 days for p24 analysis and fresh supernatant and drugswere added. We noted that no cytotoxic effects were observed in thisexperiment (also see Table 6). The results of these experiments arepresented in FIG. 2.

[0095] Our results show that concentrations of hydroxyurea as low as0.05 mM blocked HIV-1 replication (>99.9%). Use of lower doses ofhydroxyurea and AZT, at concentrations at which each of the two drugswere only partial effective, resulted in complete inhibition (>99.9%).The synergistic effects of hydroxyurea and AZT that were observed inmacrophages were therefore consistent with the results obtained usingprimary PBL treated with hydroxyurea and ddl.

[0096] Our demonstration that hydroxyurea inhibited two different HIV-1strains in primary human cells suggested this drug, either alone or incombination, could also be effective in vivo. To further test thispossibility we confirmed the previous observations by employing anotherin vitro system for drug testing. This in vitro system made use ofprimary cells isolated from HiV-1-infected individuals. We believe thismodel of HIV-1 inhibition closely approximates in vivo conditions, sinceit combines the use of primary cells and viral isolates, in the settingof an infection that was established in vivo.

[0097] Example 7 illustrates that hydroxyurea, either alone or incombination with nucleoside analogs, inhibits HIV-1 replication in cellsisolated directly from an HIV-1 infected patient.

EXAMPLE 7 Inhibition of HIV-1 in Activated PBL from an HIV-1 InfectedPatient

[0098] PBL were isolated and stimulated for 2 days with PHA and IL-2.Subsequently, bydroxyurea and ddl were added at the specifiedconcentrations. The extent of HIV-1 infection was analyzed as describedin Example 5. Samples were tested for (a) p24 production in thesupernatant, (b) viable cell count.

[0099] Once again, hydroxyurea inhibited HIV-1 replication in adose-dependent manner and, in combination with ddl, showed strongsynergistic effects (FIG. 3a). However, in some instances, both thepharmacologic and the cytotoxic effects of hydroxyurea were morepronounced (FIGS. 3a, 3 b), and lower doses of hydroxyurea (compared tothe experiments on PBL derived from healthy donors and illustrated inFIG. 1 were used, especially with 6ells from HIV-1-infected patients inthe advanced stages of AIDS. Also note that at the lowest levels bothhydroxyurea and ddl in some cases (as illustrated in FIG. 3) stimulatedHIV-1 replication, but only when used individually.

[0100] This phenomenon was not confined to the use of cells frominfected patients, since it was also occasionally observed when cellsfrom a healthy donor were used. Independent of the viral or cellularsource, however, stimulatory effects were not observed when hydroxyureaand either of the nucleoside analogs were used in combination (notshown).

[0101] Example 8 illustrates the effect of high doses of hydroxyurea onHIV-1 infection of activated PBL and macrophages that were isolated fromhealthy donors.

EXAMPLE 8 The Effect of High Concentrations of Hydroxyurea on HIV-1Infection

[0102] Experiments were conducted as described in FIGS. 1 (for PBL) and2 (macrophages) with 1 mM hydroxyurea. Percentages of HIV-1 inhibitionwere calculated based on p24 production compared to the untreatedcontrol. Drug treatment of macrophages was suspended after 14 days. Theresults of this experiment are presented in Table 6. TABLE 6 1 mMHydroxyurea drug suspension no drug Days after infection 4 7 10 14 21 2835 4 7 10 14 21 28 35 PBMC HIV-1 inhibition, % 100 100 100 100 n.d. nd.nd. 0 0 0 0 n.d n.d n.d Viable cells, 500 185 87 36 n.d. n.d. n.d. 500610 1500 1300 n.d n.d n.d thousands/ml Macrophages HIV-1 inhibition, %100 100 100 100 100 100 100 0 0 0 0 0 0 0 Viable cells, 300 280 270 270260 240 190 300 310 310 290 270 230 200 thousands/cm²

[0103] Continuous treatment with 1 mM hydroxyurea completely blockedHIV-1 replication both in activated PBL and macrophages. In activatedPBL, however, toxic effects at these concentrations were observed early,in contrast with the lack of significant toxicity in macrophages.Furthermore, in some experiments the absence of HIV-1 replication ininfected macrophages was documented even several weeks afterdiscontinuing the drug treatment.

[0104] Our finding that hydroxyurea, alone or in combination withnucleoside analogs, efficiently inhibited HIV-1 replication in primaryhuman cells in vitro suggests this drug will also be useful in humantherapy.

[0105] Example A9 describes the use of hydroxyurea in a protocoldesigned to control in vivo HIV-1 replication, thereby benefitting thetreated individual.

EXAMPLE 9 Administration of Hydroxyurea to HIV Infected Humans

[0106] One or more HIV-1 seropositive volunteers are first identified.Blood samples drawn from the volunteers are assayed for CD4⁺T-cellsusing any suitable quantitation means. Such quantitation means include,but are not limited to, the flow cytometer. Over a period of fromseveral weeks to months, the number of CD4⁺T-cells is observed todecrease steadily as an indicator of disease progression.

[0107] The HIV-1 infected volunteers are then put on a regimen of drugtherapy that includes hydroxyurea, either alone or in combination withnucleoside analogs. The nucleoside analogs can be any of ddl, ddC orAZT, or combinations thereof.

[0108] Hydroxyurea is combined with a pharmaceutically acceptableexcipient and is administered in dosages of from 20-40 mg per kg perday. The drug dosage is adjusted to result in a stable hydroxyurea bloodconcentration of approximately 1 mM. This concentration is chosenbecause it approximates the blood concentration of hydroxyurea duringstandard clinical protocols in humans. When hydroxyurea is used inconjunction with a nucleoside analog, the dosage of the analog isdetermined according to convention in the medical and pharmaceuticalarts.

[0109] After one month of drug treatment blood samples are again drawnand assayed for CD4⁺T-cells. The T-cell population has stabilized orincreased as an indication of the therapeutic effectiveness of theantiviral activity of hydroxyurea.

[0110] The most dramatic improvements are observed in volunteers whoreceived the combination of hydroxyurea together with a nucleosideanalog.

[0111] The preceding Examples have presented results obtained usingcombinations of hydroxyurea and certain chain-terminating compounds,such as nucleoside analogs, to inhibit reverse transcriptase dependentviral replication. We also expect the chain-terminating efficiency ofother dideoxynucleoside phosphate analogs, and derivatives thereof, tobe enhanced by combination drug therapy involving hydroxyurea. Hence,fluorinated derivatives of purine dideoxynucleosides, such as thosedescribed by Marquez et al. in J. Med. Chem. 33:978-985 (1990), areexpected to exhibit particularly potent antiviral activities whenadministered in combination with hydroxyurea. These fluorinatedderivatives include 2′-F-dd-ara-A, 2′-F-dd-ara-I and 2′-F-dd-ara-G.Advantageously, such fluorinated derivatives are expected to be usefulas oral medications because of their chemical stability under acidicconditions.

[0112] Example 9 describes an experiment that can be used to assess thein vitro anti-viral effects of hydroxyurea and various fluorinatedderivatives of chain terminating nucleoside analogs.

EXAMPLE 9 Use of Hvdroxvurea and Fluorinated Derivatives of ChainTerminating Nucleoside Analogs to Inhibit HIV Expression

[0113] PBL isolated from healthy donors are stimulated with PHA and IL-2for 48 hours using standard protocols. At the same time, the cells arepre-treated with hydroxyurea alone or in combination with either ddl orfluorinated derivatives of chain-terminating nucleosides. The use of ddlin this procedure serves as a positive control for hydroxyurea-enhancedinhibition of p24 production. At the end of the 48 hour period, samplesof the treated cells are infected with HIV-1 IIIB (Popovic et al.,supra). Aliquots of the cell supernatants are then harvested at varioustime points post-infection and analyzed for the presence of p24 antigenas an indicator of HIV-1 infection. Example results expected in thisprocedure are qualitatively presented in Table 7. TABLE 7 Viralexpression of p24 protein after HIV-1 infection of PHA-stimulated PBL inthe presence of μM of hydroxyurea (HU) and/or nucleoside analogs p24Expression after Infection (Days) Treatment 4 8 12 Untreated Low HighVery High HU Low Medium High 50 ddl Low Medium Medium 20 2′-F-dd-ara-ALow Medium Medium 20 2′-F-dd-ara-I Low Medium Medium 20 2′-F-dd-ara-GLow Medium Medium 20 HU + ddl Low Low Low 50 + 20 HU + 2′-Fdd-ara-A LowLow Low 50 + 20 HU + 2′-F-dd-ara-I Low Low Low 50 + 20 HU +2′-F-dd-ara-G Low Low Low 50 + 20

[0114] Results such as those presented.in Table 7 will confirm that theantiviral activities of fluorinated chain-terminating nucleoside analogsare enhanced when used in combination with hydroxyurea.

[0115] We have demonstrated that hydroxyurea is an effective HIV-1inhibitor. Significantly, these antiviral properties were not solelymediated by the cytostatic or cytotoxic effects of the drug innon-stimulated PBL and macrophages. We believe this was true becausethese cells were either quiescent (PBL) or terminally differentiated(macrophages), and therefore did not require high levels of dNTPsynthesis. Even after PBL activation, when dNTP synthesis was requiredfor cell cycling, the antiviral and cytotoxic effects could bedistinguished at low drug concentrations. The selective anti-HIV-1activity of hydroxyurea in activated PBL may be partly explained by thedistributive properties of HIV-1 RT. Compared to cellular polymerases,the distributive property of RT may render it more sensitive to lowintracellular concentrations of dNTP. In activated PBL, the cytostaticproperties of hydroxyurea probably contributed to its antiviralactivity, since viral replication in lymphocytes requires cell division.

[0116] By decreasing the intracellular concentration of dNTP whileincreasing the uptake and metabolism of nucleoside analogs, such as ddlor AZT, hydroxyurea decreased the ratio between intracellular dNTP andnucleoside analogs, thus enhancing their antiviral effects.

[0117] Combinations of hydroxyurea and either ddl or AZT proved to beextremely effective antiviral treatments. In particular, thiscombination decreased the drug concentrations necessary to obtain >99.9%inhibition of HIV-1 replication, and gave clear synergistic effects overthe use of the individual drugs without increasing their cytotoxicities.The phenomenon of viral stimulation that is sometimes observed when lowdosey of drugs are used individually was also eliminated. The combineduse of these drugs may therefore be beneficial and safe forasymptomatic, seropositive individuals.

[0118] The use of hydroxyurea in the treatment of AIDS offers severaladvantages. After more than 30 years in human use, the properties ofthis drug are well established. As a result of its extreme diffusiblity,this drug can enter all tissues, including cells of the central nervoussystem, with a V_(max) that appears infinite (Morgan, J. S., Creasey, D.C. and Wright, J. A., Biochem. Biophys. Res. Commun. 134:1254 (1986)).In view of the fact that hydroxyurea is highly effective at inhibitingHIV-1 replication in macrophages, we expect this drug to be effectiveagainst the neurological manifestations of AIDS, which are believed dueto the effects of viral replication in macrophages (Koenig, S., et al.,Science 233:1089 (1986)).

[0119] The activity of hydroxyurea does not depend on the metabolism ofthe drug within cells. Thus, in contrast with nucleoside analogs,hydroxyurea is expected to be effective in all cells, independent oftheir activation state. Hydroxyurea is classified as a mildly toxic drugand does not cause immunodepression. Myelotoxicity is hydroxyurea'sdose-limiting toxicity. However, such toxicity can be easily monitoredand it is constantly and rapidly reversible after decreasing the dose orsuspending the treatment (Donehower, R. C., Semin. OncoL 19:11 (1992)).By monitoring simple parameters like peripheral cell counts, hydroxyureacan be administered for years, and sometimes decades. Furthermore, bonemarrow toxicity is severe only when hydroxyurea is used at very highdoses, such as those used in leukemia treatment (approximately 0.5-2.5mM) (Belt, R. J. et al., Cancer 46:455 (1980)). In most of ourexperiments, hydroxyurea concentrations that were 2-3 logs lower thanthese levels still were adequate to completely inhibited HIV-1replication. Hydroxyurea can be orally administered and is much lessexpensive than other drugs that are presently used for AIDS therapy.Hydroxyurea does not inhibit HIV-1 directly, but via the inhibition ofthe cellular enzyme ribonucleotide reductase. Cellular enzymes do notmutate under physiological conditions and one could expect that HIV-1resistance to hydroxyurea would be far less likely to occur than withconventional drugs. This could circumvent the onset of HIV-1 escapemutants. To date, none of the anti-HIV-1 drugs that have been testedhave prevented the evolution of escape mutants. This failure representsa major frustration in the battle against AIDS. Moreover, the onset ofescape mutants that arise during treatment of AIDS victims withnucleoside analogs, should also be reduced when these drugs are used incombination with hydroxyurea. Since the synergistic effect of thecombination of a nucleoside analog and hydroxyurea inhibits virusreplication, which may be a requisite step in the process of virusmutation that leads to the development of escape mutants.

[0120] In our opinion, two main strategies utilizing hydroxyurea as AIDStherapies may be followed. The first is the use of low doses ofhydroxyurea. Drug combinations are recommended in this case, for thereasons above illustrated, and trials could safety include asymptomaticseropositive individuals. The second strategy would use high levels ofhydroxyurea, with protocols similar to those used in leukemia. Thisstrategy would be more potent against HIV-1 and would also kill thereplicating PBL producing virus. However, one could design a combinationof both strategies by alternating high doses of hydroxyurea for purgingpurposes, followed by lower maintenance doses.

[0121] While particular embodiments of the invention have been describedin detail, it will be apparent to those skilled in the art that theseembodiments are exemplary rather than limiting, and the true scope ofthe invention is that defined by the claims that follow.

We claim:
 1. A method for inhibiting replication of reversetranscriptase dependent virus in animal cells, comprising the step ofadministering to said cells a compound that depletes the intracellularpool of deoxyribonucleoside phosphate in an amount effective to inhibitreplication of said virus.
 2. The method of claim 1 , wherein said virusis a retrovirus.
 3. The method of claim 1 , wherein said deoxynucleosidephosphate depleting compound is a deoxynucleotide synthesis inhibitor.4. The method of claim 1 , wherein said deoxynucleoside phosphatedepleting compound is an inhibitor of ribonucleotide reductase.
 5. Themethod of claim 4 , wherein said compound is hydroxyurea.
 6. The methodof claim 1 , wherein said cells are in vitro.
 7. The method of claim 1 ,wherein said animal cells are mammalian cells.
 8. The method of claim 1wherein said virus is the human immunodeficiency virus (HIV) and saidcells are human cells.
 9. A method for inhibiting replication of reversetranscriptase dependent virus in animal cells, comprising the steps ofadministering to said cells a compound that depletes the intracellularpool of deoxyribonucleoside phosphate, in conjunction with administeringto said cells an antiviral nucleoside phosphate analog.
 10. The methodof claim 9 , wherein said deoxynucleotide phosphate depleting compoundis an inhibitor of ribonucleotide reductase.
 11. The method of claim 10, wherein said compound is hydroxyurea.
 12. A method for inhibitingreplication of reverse transcriptase dependent viruses in animal cells,comprising the steps of administering to said cells a first compoundthat depletes the intracellular pool of deoxyribonucleoside phosphate,in conjunction with a second compound that serves to inhibit replicationof said virus by terminating DNA chain elongation.
 13. The method ofclaim 12 , wherein said second compound inhibits replication bypremature termination of viral DNA synthesis to produce incomplete viralDNA.
 14. The method of claim 12 , wherein said first compound is aninhibitor of ribonucleotide reductase.
 15. The method of claim 14 ,wherein said first compound is hydroxyurea.
 16. The method of claim 15 ,wherein said second compound is selected from the group consisting ofddl, ddC, 2′-F-dd-ara-A, 2′-F-dd-ara-I and 2′-F-dd-ara-G.
 17. The methodof claim 12 , wherein said second compound is selected from the groupconsisting of a dideoxynucleoside and AZT.
 18. The method of claim 1 6,wherein said dideoxy nucleoside is a 2′-fluoro purine dideoxynucleoside.19. The method of claim 16 , wherein said dideoxynucleoside is selectedfrom the group consisting of ddl, ddC, 2′-F-dd-ara-A, 2′-F-dd-ara-I and2′-F-dd-ara-G.
 20. A method of producing incomplete viral DNA from areverse transcriptase dependent virus in animal cells, comprising thestep of administering to said cells a compound that depletes theintracellular pool of deoxyribonucleoside phosphate in an amounteffective to inhibit replication of said virus.